Heart failure is a widespread phenomenon, especially in the western world. According to the Roche Medical Dictionary (ed. Hoffmann-La Roche A G, Urban & Schwarzenberg, Munich, 1993), heart failure is the acute or chronic inability of the heart to generate the blood flow required for metabolism during exercise or even at rest or to assure the venous reflux (backward and forward failure). Thus the pump function of the heart is weak. The causes of heart failure are very complex. Among others, inflammatory and degenerative modifications of the cardiac muscle, coronary perfusion disorder, coronary infarction and injuries are to be mentioned here. This leads to modifications of the peripheral bloodstream, disorders of the breathing system, renal function, and electrolyte metabolism (edema,) and to a reduced performance of the muscular system of the skeleton.
According to the New York Heart Association (NYHA), heart failure is divided into the following NYHA classes using physical tests after effort: I means completely free from pain after normal physical effort, II means low limitation of the physical toughness, III means strong limitation of the physical toughness, IV means that with each physical activity, the insufficiency symptoms increase which most of the time also exist at rest.
For an effective medicament treatment of heart failure by means of glycosides, vasodilators, ACE inhibitors, and/or β-blockers, it is first of all necessary to exactly and correctly identify and diagnose heart failure, to classify it, if possible, according to the degree of severity, and to additionally monitor the course of treatment.
In the art, some serum markers are discussed as marker candidates for an early diagnosis of heart failure as, for example, ANP (atrial natriuretic peptid) hormone and proANP, CNP (C-natriuretic peptide), adrenomedullin, neuropeptide Y, endotheline, and BNP (brain natriuretic peptide). ANP and proANP theoretically would represent suitable markers for the diagnosis of heart failure; in practice they are, however, not very stable or only have a short half life in blood, which represents a serious drawback to routine diagnostic measurements (Buckley, M. G., et al., Clin. Sci. 95 (1998) 235–239; Cleland, J. G., et al., Heart 75 (1996) 410–413).
A frequently cited and meaningful marker is BNP (brain natriuretic peptide). Originally, BNP was identified in the brain of pigs. It is a cardiac hormone which structurally and functionally resembles ANP (Sudoh, T., et al., Nature 332 (1988) 78–81). Human BNP, consisting of 32 amino acids, is mainly secreted by the heart ventricles and circulates in the human blood plasma. The use of BNP as a diagnostic marker is, for example, known from EP 542,255. BNP has an intramolecular disulfide bridge and is not a very stable analyte. This presumably is due to its physiological function as a hormone that must be broken down quickly. Therefore, its use as a diagnostic marker requires careful and special attention in sample collection and processing (Masuta, C., et al., Clin. Chem. 44 (1998) 130; Tsuji, T., et al., Clin. Chem. 40 (1994) 672–673).
The precursor molecule of BNP, i.e., proBNP, consists of 108 amino acids. proBNP is cleaved into the aforementioned 32 C-terminal amino acids (77–108) called BNP and the N-terminal amino acids 1–76 called N-terminal proBNP (or NT-proBNP). BNP, N-terminal proBNP (1–76) as well as further breakdown products (Hunt, P. J., et al., Biochem. Biophys. Res. Com. 214 (1995) 1175–1183) circulate in blood. Whether the complete precursor molecule (proBNP 1–108) also occurs in the plasma is not completely resolved. It is, however, described (Hunt, P. J., et al, Peptides, Vol. 18, No. 10 (1997), 1475–1481) that a low release of proBNP (1–108) in plasma is detectable but that, due to the very quick partial breakdown at the N-terminal end, some amino acids are absent.
As known from the art, the N-terminal proBNP (1–76) is considered a marker of heart failure.
WO 93/24531 and U.S. Pat. No. 5,786,163 describe an immunological method of identifying N-terminal proBNP and the antibodies used for it. In WO 93/24531, polyclonal antibodies (PAB's) against one single peptide derived from the N-terminal proBNP are produced. It is shown that the antibodies produced bind to the immunization peptide (amino acids 47–64) in a competitive test format.
In the competitive test performed in WO 93/24531, the peptide 47–64 in a labelled form competes as a tracer with proBNP in a sample or the unlabelled peptide standard 47–64 for binding to polyclonal antibodies from rabbit serum. Only a moderate competition is reached after 48 hours of incubation, resulting in a lower limit of detection of approximately 250 fmol/ml. The long incubation times of this competitive test are not acceptable for routine measurements of the samples in automated laboratories.
Hunt, P. J., et al., Clinical Endocrinology 47 (1997) 287–296, also describe a competitive test for the detection of N-terminal proBNP. In this assay, a complex extraction of the plasma sample is necessary before the measurement can be performed; this may lead to the destruction of the analyte and erroneous measurements. The antiserum used is produced analogously to WO 93/24531 by immunization with a synthetic peptide. Hunt et al. produce the antiserum by immunization with the N-terminal proBNP amino acids 1–13, and a peptide consisting of amino acids 1–21 is used as a standard. For this test, long incubation times are necessary, too. After an incubation of 24 hours, a lower detection limit of 1.3 fmol/ml is reached.
Ng, L., et al., WO 00/35951 describe a further method for determining N-terminal proBNP. This method is based on use of antibodies raised against a synthetic peptide corresponding to amino acids 65 to 76 of human proBNP.
Hughes, D., et al., Clin. Sci. 96 (1999) 373–380, report on two different assays for N-terminal proBNP. In a first assay, a polyclonal antibody generated with an immunogen comprising a synthetic peptide corresponding to amino acids 65–76 of proBNP is used, whereas in a second assay, the polyclonal antibody was generated in analogy but to amino acids 37–49. According to the data produced by Hughes, D., et al., an antibody generated and reactive with the peptide corresponding to amino acids 37–49 of proBNP does not react with intact endogenous proBNP. An assay based thereon does not discriminate patients with left ventricular dysfunction from normal controls. With the assay based on proBNP 65–76, the same patient groups could be clearly discriminated.
Goetze, J. P., et al., Clin. Chem. 48 (2002) 1035–1042, describe an assay for the most N-terminal amino acids (1–21) of N-terminal proBNP. Their assay is based on a polyclonal antibody raised against a synthetic peptide corresponding to the same amino acids (1–21) of proBNP.
The assay of Goetze, J. P., et al., supra, requires complete digestion of the sample and the various proBNP's comprised therein. It is said that this assay also was efficient in reduction of non-specific binding.
Karl, J., et al., WO 00/45176, for the first time show that sensitive and rapid detection of N-terminal proBNP is possible in a sandwich immunoassay. Preferred epitopes, as described in WO 00/45176, are between amino acids 10 and 50 of N-terminal proBNP.
US 2003/0219734 refers to the fact that a plurality of different BNP-related polypeptides derived from proBNP (1–108), BNP (77–108), as well as from N-terminal proBNP (1–76) may be present in a sample.
Mair, J., et al., Clin. Chem. Lab. Med. 39 (2001) 571–588, have summarized the impact of cardiac natriuretic peptide determination on the diagnosis and management of heart failure. They stress that currently available commercial assays are not standardized, i.e., they have not been calibrated against common standards. In some assays even an extraction of plasma is needed. Consequently, the results obtained with assays from different manufacturers may differ markedly. Therefore, reference intervals and decision limits derived from clinical studies are only valid for the particular assay used and must not be extrapolated to other assays for N-terminal proBNP.
Along the same lines, Goetze, J. P., et al., supra and Mair, J., Clin. Chem. 48 (2002) 977–978, note that the discrepancies between different assays of N-terminal proBNP, both with respect to the values obtained as well as with regard to their clinical implications, represent a crucial problem to the widespread use of this marker candidate.
Obviously a great need exists to provide for an improved, e.g., more reproducible, better standardized, better characterized, and more clinically relevant assay for N-terminal proBNP.
It was a task of the present invention to develop a more specific assay for measurement of N-terminal proBNP and/or a clinically relevant fragment or subpopulation of N-terminal proBNP.
The invention as described below and claimed in the appending claims at least partially solves one or more of the problems known in the art.